Current DNA sequencing technology has made it both inexpensive and rapid to sequence whole genomes for a variety of important applications. The amount of available sample DNA, however, is often highly limited, thus requiring amplifying the DNA before sequencing. Genome amplification techniques generally utilize DNA polymerases that are capable of isothermally displacing DNA strands in concert with the DNA polymerization process.
The two most widely used strand displacing enzymes for DNA amplification are bacteriophage Phi29 (also denoted as Φ29) DNA polymerase (“DNApol”) and BstI (also referred as Bst) DNA polymerase. These two enzymes are currently the gold standard for isothermal nucleic acid amplification.
The Φ29 DNA polymerase was used to develop multiply-primed rolling circle amplification using random hexamer primers. The Φ29 DNApol, a family B member, has high processivity, high fidelity, comparable to the best enzymes commercially available for PCR, due to its “proofreading” 3′-5′ exonuclease activity, and significant strand displacement activity. Although not thermostable for PCR, the Φ29 DNA polymerase has a broad operational temperature range, extending down into typical room temperatures, allowing reactions to be carried out at ambient temperature. Recently, through protein domain “swapping and tagging,” the Φ29 DNA polymerase has been engineered for increased processivity. The Φ29 DNApol activity, however, is not very tolerant of high ionic strength.
The family A member BstI DNA polymerase large fragment lacks intrinsic 3′-5′ exonuclease activity; thus, its fidelity is significantly less than that of the Φ29 DNApol. The large fragment of the Bst DNApol is prepared by partial proteolysis of the native enzyme with subtilisin, which cleaves the 5′ exonuclease moiety. BstI DNApol is thermostable, so that DNA amplification reactions can be carried out at 65° C., which may be useful for amplifying GC-rich targets, and it is possible to carry out sequential extension and melting reactions desirable for some applications. However, BstI DNA polymerase activity at 37° C. is 10-15% of its activity at 65° C. and it is inactivated by heating at 80° C. for 20 minutes. BstI DNApol large fragment activity is intolerant of ionic strength above 60 mM KCl; however, recently, engineered forms of BstI DNA polymerase have become available that will tolerate ionic strength up to 150 mM KCl.
Examples of published patent applications and issued patents related to DNA polymerases and their use in amplification of nucleic acid sequences include EP Publication No. EP 07112927; EP Publication No. EP 2210941; U.S. Pat. No. 5,576,204; and U.S. Pat. No. 6,124,120, the disclosures of each of which is incorporated by reference herein.
The current gold standard method for whole-genome amplification (“WGA”) employs the error-prone BstI DNA polymerase, with an error rate of 10−4. This method, Multiple Looping-Based Amplification Cycles (“MALBAC”), only allows amplification of, at best 93% and less in many cases, of the human genome efficiently, for single-cell genome sequencing. The high error rate and the inability to amplify 100% of the genome are major technical challenges and significant disadvantages of this method.
Most DNA copying enzymes have a mis-incorporation (error) rate of 10−4, unless they have an intrinsic proofreading 3′-5′ exonuclease, which BstI DNApol large fragment lacks. DNA polymerases with 3′-5′ exonuclease domains typically are characterized by error rates of about 10−8. On the other hand, error rates typically increase to about 10−6 for DNA polymerases with site-specific exo-mutants. Exo-mutants often have increased elongation rates and increased processivity due to diminished “idling,” and often have increased strand-displacement activities. An amplification error rate in the order of 10−8 dictates that tens of erroneous single-nucleotide changes could be introduced into an amplified single-cell genome. If single-cell genome amplification is being carried out for the purpose of detecting single nucleotide mutations, then multiple amplifications must be carried out and sequenced to show statistically that the changes are not due to the amplification method. It follows that the more error-prone the enzyme, the more error-prone the amplification method, and the greater the amount of duplication that will be necessary.
Some of the problems of amplification bias and introduction of copying errors are linked to intrinsic properties of the DNApol enzymes used to amplify the DNA to be sequenced. At present there is no single enzyme being used in these protocols with all of the properties of an ideal copier; thus there is a need for a different DNA polymerase possessing some critical characteristics. Of the many DNA polymerases that have been characterized, comparatively few possess significant strand-displacement activity (“SDA”), one characteristic necessary for WGA applications. The linkage of SDA to processivity and proofreading 3′-5′ exonuclease activity appears also to be important.
Another desirable property for a DNA polymerase for use in amplifying nucleic acids in vitro (such as single-cell genome amplification) is tolerance (i.e., ability to function) of a broad range of salt concentrations (“salt tolerance”). Salt tolerance of an enzyme may allow considerable latitude and flexibility for designing new priming strategies, since priming involves hybridization, and hybridization conditions are highly salt sensitive.